The PI has made significant advances in her research and drug education activities to the present time, and has the potential to answer significant questions as to the mechanism of tolerance and physical dependence if given a sustained commitment by NIDA to the goals of this research. Specifically, the objective of this research is to determine the role of free intracellular calcium and cyclic-AMP in the auction of antinociception, tolerance, and physical dependence by two classes of drugs, opiates and cannabinoids. Opiate tolerance is accompanied by increased levels of intracellular free calcium measured directly with the indicator fura-2/AM. Some possible mechanisms for this rise include modulation of calcium channel activation or number, potassium channel modulation, activation of c-AMP, or the influence of endogenous calcium modulators. These possibilities will be examined in both synaptosomes and dorsal root ganglia (DRG). In addition, we hypothesize that the chronic administration of opiates alters the functionality, specific binding, or mRNA for the endogenous calcium modulatory peptides calcitonin (CT) and calcitonin gene-related peptide (CGRP) leading directly to compensatory modulation of intracellular calcium in neurons or indirectly via activation of adenylyl cyclase. This hypothesis will be examined by the use of receptor binding assay for the hormones, radioimmunoassays, autoradiography, immunohistochemistry, and the measurement of c-AMP accumulation in synaptosomes from brain regions and the spinal cord. The novel profile of pharmacological activity of the cannabinoids indicates that these drugs may be useful tools in the elucidation of what may prove to be a unique mechanism for the relief of pain. Our data indicate that the antinociceptive effects of the cannabinoids occur due to interaction at both supraspinal and spinal sites, although the mechanism of action of the drugs is not known. Using methods similar to those described for the opiates, we propose to perform a systematic study of the role of calcium and cyclic-AMP in the antinociceptive effects of the cannabinoids in order to compare and contrast the mechanism of action of the drugs in the brain versus in the spinal cord using synaptosomes and DRG cells. We have shown that the cannabinoids significantly potentiate the antinociceptive effects of opiates at spinal and supraspinal site. In addition, we have found that the antinociceptive effects of cannabinoids are blocked by the kappa opiate antagonist, nor-BNI, but not mu and delta receptor blockers. We hypothesize that the mechanism of action of the cannabinoids may have similarities to the opiates in the alteration of neuronal second messengers. We propose to determine the interaction of the cannabinoids and the opiates with intracellular calcium and c-AMP production. The outcome of all of these studies could lead to a better understanding of the process of addiction and interventions in the addiction process.